HPLC Columns for Reliable, Reproducible Method Development

Types of HPLC Columns: Which Column Should You Choose?

HPLC columns are available in a wide range of types, each designed to address specific analytical challenges depending on the nature of compounds to be separated. Understanding the different separation mechanisms helps ensure you select the column that delivers optimal retention, selectivity, and resolution for your analysis.

How to Choose a Column for HPLC

The column is the most important aspect for all separations, as this is where the stationary phase resides, and provides the main separation mechanism. The stationary phase’s tightly packed bed is enclosed/encased in a cylindrical space/tube of a specific length and internal diameter. Most columns are made out of stainless steel, while PEEK (polyetheretherketone) or glass are also used. Inert surfaces may also be employed for specific separations that are very sensitive to the column’s material where interactions can also occur.

The selection of an HPLC column, amongst all the different HPLC column types is a critical set of decisions that can significantly impact the retention, efficiency, selectivity and hence the resolution as well as the reproducibility of the separation process.

Key factors during the column selection decision include:

Particle Size / Particle Technology

• Efficiency: The particle size of the stationary phase material directly affects the column’s efficiency/performance. Smaller particles were developed to provide a higher number of theoretical plates (N) at the expense of increased backpressure and suffer from frictional heating peak dispersion/broadening effects. 
• Columns with sub-2 µm particles must be employed and used with instrumentation that can be operated at ultra-high-pressure environments (>600 bar and up to 1500 bar) - this specific technique is known as ultra high pressure liquid chromatography (UHPLC). 
• While smaller particles enhance efficiency, they require knowledge and expertise to exploit their benefits and understand the theoretical and practical drawbacks of using ultra high pressures for their separations. For example, understanding frictional heating peak broadening/dispersion effects. Also, to take into account how they are packed with smaller sized frits that are sensitive to blockage and/or contamination, hence, require very clean laboratory practices with sample and mobile phase preparation. 
• Particle technology: The most commonly used type of particle are fully porous particles (FPP). Superficially porous particles (SPP), also known as solid-core, core-shell, pellicular particles, are an alternative particle that have practical benefits of increased efficiency at lower backpressures relative to FPP of the same size.
• Particle technology: The most commonly used type of particle are fully porous particles (FPP). Superficially porous particles (SPP), also known as solid-core, core-shell, pellicular particles, are an alternative particle that have practical benefits of increased efficiency at lower backpressures relative to FPP of the same size.

Column Dimensions / Format

• Length (L): The length of the column is directly related to the column efficiency/performance associated to the metric known as the number of theoretical plates (N). Typical lengths are between 5-25 cm. 
• Long column lengths: provide a higher N, increased column performance, at the expense of increased analysis time due to the increase of the column’s dead time (T0) contribution (an important metric that the analyst must also be familiar with). Increasing L, increases solvent consumption, and back-pressure. They are suitable for complex mixtures where maximum column performance is necessary. 
• Reduction of column length: this is acceptable as long as column efficiency remains sufficient for the separation aims/objectives/goals. Reducing L, reduces T0, and may decrease the analysis time and back pressure. 
• Diameter: The internal diameter (ID) of the column is selected based on the separation’s sample injection volume, loading capacity, velocity/flowrate and sensitivity. The typically employed ID dimension is between 2.1 and 4.6 mm for analytical scale separations. Wider bore columns >4.6 mm ID allow for larger sample volumes. These IDs are good options for purification applications, where large injection volumes are employed. 
• ‘Narrow-bore’ columns of 2.1 mm or smaller are used for applications with limited sample volumes and/or concentrations. Employed to increase the detection sensitivity of the analysis with a reduced injection volume. 
• Reducing the ID of a column lead to a loss of column performance due to the column wall effect becoming a larger contributing factor to peak dispersion/broadening effects.

Temperature and Flow Rate

• Temperature: Column temperature can enhance separation efficiency. Increasing the temperature is highly beneficial for reducing the solvent viscosity, allowing for faster flow rates to be used at reduced backpressure. Increasing the mass transfer kinetics between the analytes and the mobile and stationary phase. It is one of the most underutilized practical parameters that can easily change the selectivity, efficiency and retention of the separation, hence the resolution. Analysts must ensure that the analytes and stationary phase and system are thermally stable/compatible under the chosen elevated temperature conditions by checking with the manufacturers’ operational limits. 
• Flowrate: The flowrate/velocity of the mobile phase is tightly coupled to the performance of the column. The classical and historical Van Deemter curve experiments highlights this dependency and is also another parameter underutilized, not many laboratories use the Van Deemter curve minimum/optimum velocity for their flow rate selection. Often use flowrates based on how long can they can afford to spend on each injection, or use the flowrate associated to the certificate of analysis of the purchased column. Higher flow rates reduce analysis time but may compromise separation column performance and increase backpressure, particularly with UHPLC columns. Also, we must note that detectors such as mass spectrometry (MS) as well as evaporative light scattering and charged aerosol detectors are sensitive to volumetric flowrate changes, hence, if the detection techniques are prioritized, this is also an aspect that will effect the selection of an appropriate flowrate and column format.

Stationary Phase Chemistry

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Particle

• Selectivity/bonded phase/bonded chemistry/bonded moeity/bonded ligand: Selectivity in general is the key approach to achieving higher resolution separations. The most common way is to change the mobile phase selectivity, and the easiest approach is to change to change the temperature. However, the parameter that is highly impactful is to change the HPLC column stationary phase chemistry. 
• Analytes are often separated based on their hydrophobic characteristics via reversed phase (RP) columns with hydrophobic bonded chemistries. Analytes can also be separated based on their polarity via normal phase (NP) columns with polar stationary phase chemistries. Very polar analytes may be separated using hydrophilic interaction liquid chromatography (HILIC) stationary phase columns. 
• RP columns are the most employed and the C18 column has become one of the most utilized columns in validated methods. We must not ignore that there are other interactions/mechanisms that can be exploited to improve the selectivity and resolution of the separation. Alternative/specialized stationary phases are available for unique or more complex separation challenges, such as chiral separations or the analysis of highly polar or ionic compounds.